Jun N-Terminal Kinase (JNK)
The Jun N-terminal kinase (JNK) pathway is activated by exposure of cells to environmental stress or by treatment of cells with pro-inflammatory cytokines and growth factors. Targets of the JNK pathway include the transcription factors c-jun and ATF2 (Whitmarsh A. J., and Davis R. J. J. Mol. Med. 74:589-607, 1996). These transcription factors are members of the basic leucine zipper (bZIP) group that bind as homo- and hetero-dimeric complexes to AP1 and AP-1-like sites in the promoters of many genes (Karin M., Liu Z. G. and Zandi E. Curr Opin Cell Biol 9:240-246, 1997). JNK binds to the N-terminal region of c-jun and ATF-2 and phosphorylates two sites within the activation domain of each transcription factor (Hibi M., Lin A., Smeal T., Minden A., Karin M. Genes Dev. 7:2135-2148, 1993; Mohit A. A., Martin M. H., and Miller C. A. Neuron 14:67-78, 1995). Three JNK enzymes have been identified as products of distinct genes (Hibi et al, supra; Mohit et al., supra). Ten different isoforms of JNK have been identified. These represent alternatively spliced forms of three different genes: JNK1, JNK2, and JNK3. JNK1 and 2 are ubiquitously expressed in human tissues, whereas JNK3 is selectively expressed in the brain, heart, and testis (Dong, C., Yang, D., Wysk, M., Whitmarsh, A., Davis, R., Flavell, R. Science 270:1-4, 1998). Gene transcripts are alternatively spliced to produce four-JNK1 isoforms, four-JNK2 isoforms, and two-JNK3 isoforms. JNK1 and 2 are expressed widely in mammalian tissues, whereas JNK3 is expressed almost exclusively in the brain. Selectivity of JNK signaling is achieved via specific interactions of JNK pathway components and by use of scaffold proteins that selectively bind multiple components of the signaling cascade. JIP-1 (JNK-interacting protein-1) selectively binds the MAPK module, MLK→JNKK1→JNK. It has no binding affinity for a variety of other MAPK cascade enzymes. Different scaffold proteins are likely to exist for other MAPK signaling cascades to preserve substrate specificity.
JNKs are activated by dual phosphorylation on Thr-183 and Tyr-185. JNKK1 (also known as MKK 4) and JNKK2 (MKK7), two MAPKK level enzymes, can mediate JNK activation in cells (Lin A., Minden A., Martinetto H., Claret F.-Z., Lange-Carter C., Mercurio F., Johnson G. L., and Karin M. Science 268:286-289, 1995; Tournier C., Whitmarsh A. J., Cavanagh J., Barrett T., and Davis R. J. Proc. Nat. Acad. Sci. USA 94:7337-7342, 1997). JNKK2 specifically phosphorylates JNK, whereas JNKK1 can also phosphorylate and activate p38. Both JNKK1 and JNKK2 are widely expressed in mammalian tissues. JNKK1 and JNKK2 are activated by the MAPKKK enzymes, MEKK1 and 2 (Lange-Carter C. A., Pleiman C. M., Gardner A. M., Blumer K. J., and Johnson G. L., Science, 260:315-319, 1993; Yan M., Dai J. C., Deak J. C., Kyriakis J. M., Zon L. I., Woodgett J. R., and Templeton D. J., Nature, 372:798-781, 1994). Both MEKK1 and MEKK2 are widely expressed in mammalian tissues.
Activation of the JNK pathway has been documented in a number of disease settings, providing the rationale for targeting this pathway for drug discovery. In addition, molecular genetic approaches have validated the pathogenic role of this pathway in several diseases. For example, autoimmune and inflammatory diseases arise from the over-activation of the immune system. Activated immune cells express many genes encoding inflammatory molecules, including cytokines, growth factors, cell surface receptors, cell adhesion molecules, and degradative enzymes. Many of these genes are regulated by the JNK pathway, through activation of the transcription factors AP-1 and ATF-2, including TNF-alpha, IL-2, E-selectin, and matrix metalloproteinases such as collagenase-1 (Manning A. M. and Mercurio F., Exp Opin Invest Drugs, 6: 555-567, 1997). Monocytes, tissue macrophages, and tissue mast cells are key sources of TNF-alpha production. The JNK pathway regulates TNF-alpha production in bacterial lipopolysaccharide-stimulated macrophages, and in mast cells stimulated through the FceRII receptor (Swantek J. L., Cobb M. H., Geppert T. D., Mol. Cell. Biol., 17:6274-6282, 1997; Ishizuka, T., Tereda N., Gerwins, P., Hamelmann E., Oshiba A., Fanger G. R., Johnson G. L., and Gelfiand E. W., Proc. Nat. Acad. Sci. USA, 94:6358-6363, 1997). Inhibition of JNK activation effectively modulates TNF-alpha secretion from these cells. The JNK pathway therefore regulates production of this key pro-inflammatory cytokine. It is believed that JNK is pro-apoptotic under stress or inflammatory conditions such as exposure to UV-radiation. (Leppa and Bohman, Oncogene 18:6158-6162 (1999)). Matrix metalloproteinases (MMPs) promote cartilage and bone erosion in rheumatoid arthritis, and generalized tissue destruction in other autoimmune diseases. Inducible expression of MMPs, including MMP-3 and MMP-9, type II and IV collagenases, are regulated via activation of the JNK pathway and AP-1 (Gum, R., Wang, H., Lengyel, E., Juarez, J., and Boyd, D., Oncogene, 14:1481-1493, 1997). In human rheumatoid synoviocytes activated with TNF-alpha, IL-1, or Fas ligand the JNK pathway is activated (Han Z., Boyle D. L., Aupperle K. R., Bennett B., Manning A. M., Firestein G. S., J. Pharm. Exp. Therap., 291:1-7, 1999; Okamoto K., Fujisawa K., Hasunuma T., Kobata T., Sumida T., and Nishioka K., Arth & Rheum, 40: 919, 1997). Inhibition of JNK activation results in decreased AP-1 activation and collagenase-1 expression (Han et al., supra). The JNK pathway therefore regulates MMP expression in cells involved in rheumatoid arthritis.
Role of JNK in Cancer and Stroke
Cancer is characterized by uncontrolled growth, proliferation and migration of cells. Cancer is the second leading cause of death with 500,000 deaths and an estimated 1.3 million new cases in the United States in 1996. The role of signal transduction pathways contributing to cell transformation and cancer is a generally accepted concept. The JNK pathway leading to AP-1 appears to play a critical role in cancer. Expression of c-jun is altered in early lung cancer and may mediate growth factor signaling in non-small cell lung cancer (Yin T., Sandhu G., Wolfgang C. D., Burrier A., Webb R. L., Rigel D. F. Hai T., and Whelan J., J. Biol. Chem. 272:19943-19950, 1997). Indeed, over-expression of c-jun in cells results in transformation, and blocking c-jun activity inhibits MCF-7 colony formation (Szabo E., Riffe M., Steinberg S. M., Birrer M. J., Linnnoila R. I., Cancer Res. 56:305-315, 1996). DNA-damaging agents, ionizing radiation, and tumor necrosis factor activate the JNK pathway. In addition to regulating c-jun production and activity, JNK activation can regulate phosphorylation of p53 and, thus, can modulate cell cycle progression (Chen T. K., Smith L. M., Gebhardt D. K., Birrer M. J., Brown P. H,. Mol. Carcinogenesis, 15:215-226, 1996). The oncogene BCR-Abl, associated with t(9,22) Philadelphia chromosome translocation of chronic myelogenous leukemia, activates JNK and leads to transformation of hematopoietic cells (Milne D. M., Campbell L. E., Campbell D. G., Meek D. W., J. Biol. Chem. 270:5511-5518, 1995). Selective inhibition of JNK activation by a naturally occurring JNK inhibitory protein, called JIP-1, blocks cellular transformation caused by BCR-Abl expression (Raitano A. B., Halpern J. R., Hambuch T. M., Sawyers C. L., Proc. Nat. Acad. Sci USA, 92:11746-11750, 1995). Thus, JNK inhibitors may block transformation and tumor cell growth.
JNK is also believed to partly responsible for cancer and/or tumor resistance to certain chemotherapeutics. The number one cause of cancers refractory against traditional chemo drugs is the upregulation of the mdr1 gene. The mdr1/p-glycoprotein gene has an AP-1 binding site in its promoter and is believed to be stimulated by JNK. Upregulation of JNK activity has also been found in tamoxifen-resistant tumors. DN-Jun inhibits tumor growth in tamoxifen-resistant animals and delays development of tamoxifen-resistant phenotype (Daschner, et al. Breast Cancer Res. 53:229, 1999; Schiff, et al. J. Natl. Cancer Inst. 92:1926, 2000).
Stroke is the 3rd leading cause of death and a leading cause of disability in the U.S. Stroke, along with neurodegenerative diseases, such as Alzheimer's (AD) and Parkinson's disease (PD) impose a huge burden on the health care industry by impacting the quality of life of those affected. Loss of neuronal cell populations in stroke, AD, or PD underlies the motor and/or cognitive deficiencies in these patient populations. The mechanism by which neurons die in response to insult has not been fully elucidated; however, activation of the JNK pathway has been implicated as a major signaling pathway for neuronal apoptosis. (For review see Mielke K. and Herdegen T. Prog. Neurobiol. 61:45-60, 2000). There have been a number of conflicting reports as to the role of JNK activity in the regulation of apoptosis. Some studies suggest that activating JNK activity induces phosphorylation of C-Jun protein and protects cells from apoptosis (Potapova, O., Basu, S., Mercola, D., Holbrook, N., J. Biol. Chem. 276:28546-28553, 2001). However, both pro-survival and pro-apoptotic roles of activated JNK activity have also been described (Kolbus, A., Herr, I., Schreiber, M., Piu, F., Beeche, M., Wagner, E. F., Karin, M., 103:897-907, 2000; Wisdom, R., Johnson, R. S., Moore, C., EMBO J., 18:1888-197, 1999). A variety of insults have been shown to activate the JNK pathway in neurons. For example, activation of JNKs and phosphorylation of c-jun has been shown in brains of rats subjected to axotomy or ischemia with reperfusion, where neuronal cell loss was observed (Herdegen T., Claret F.-X., Kallunki, T., Matin-Villalba A., Winter C., Hunter T. and Karin M. J. Neurosci. 18:5124-5135, 1998). Further, inhibition of the mixed lineage kinase (MLK)-3, an upstream kinase in the JNK pathway, by CEP-1347 prevented motor neuron cell death following growth factor withdrawal in vitro (Maroney A. C., Glicksman M. A., Basma A. N., Walton K. M., Knight Jr. E., Murphy C. A., Bartlett B. A., Finn J. P., Angeles T., Matsuda Y., Neff N. T. and Dionne C. A., J. Neurosci. 18:104-111, 1998), protected cholinergic neurons following excitotoxic injury of the nucleus basalis magnocellularis (Saporito M. S., Brown, E. R., Miller M. S., Murakata C., Neff N. H., Vaught J. L., and Carswell S. Neuroscience 86:461-472, 1998), and blocked the degeneration of midbrain dopamine neurons in mice treated with the neurotoxin, 1-methyl-4-phenyl tetrahydropyridine (Saporito M. S., Brown E. M., Miller M. S. and Carswell S. J. Pharm. Exp. Ther., 1999). While JNK1 and JNK2 enzymes have a widespread tissue distribution, JNK3 is selectively expressed in brain and to a lesser extent in the heart and testis (Dong C., Yang D., Wysk M., Whitmarsh A., Davis R., and Flavell R. Science 270:1-4, 1998). Because of this restricted distribution, JNK3 may be the prevailing kinase mediating neuronal apoptosis. In support of JNK3's involvement in neuronal apoptosis, disruption of the gene encoding JNK3 in mice confers resistance to kainic acid—induced seizures and subsequent hippocampal neuronal cell death (Yang D. D., Kuan C.-Y., Whitmarsh A. J., Rincon M., Zheng T. S., Davis R. J., Rakic P. and Flavell R. A. Nature 389:865-870, 1997). Mounting evidence points to a role for the JNK pathway in neuronal apoptosis. Therefore, selective JNK inhibitors should prevent neuronal cell death observed in disorders and diseases of the CNS.
Cancer Therapy
Currently, cancer therapy may involve surgery, chemotherapy, hormonal therapy and/or radiation treatment to eradicate neoplastic cells in a patient (see, for example, Stockdale, 1998, “Principles of Cancer Patient Management”, in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). Recently, cancer therapy could also involve biological therapy or immunotherapy. All of these approaches pose significant drawbacks for the patient. Surgery, for example, may be contraindicated due to the health of the patient or may be unacceptable to the patient. Additionally, surgery may not completely remove the neoplastic tissue. Radiation therapy is only effective when the neoplastic tissue exhibits a higher sensitivity to radiation than normal tissue, and radiation therapy can also often elicit serious side effects. Hormonal therapy is rarely given as a single agent and although can be effective, is often used to prevent or delay recurrence of cancer after other treatments have removed the majority of the cancer cells. Biological therapies/immunotherapies are limited in number and may produce side effects such as rashes or swellings, flu-like symptoms, including fever, chills and fatigue, digestive tract problems or allergic reactions.
With respect to chemotherapy, there are a variety of chemotherapeutic agents available for treatment of cancer. A significant majority of cancer chemotherapeutics act by inhibiting DNA synthesis, either directly, or indirectly by inhibiting the biosynthesis of the deoxyribonucleotide triphosphate precursors, to prevent DNA replication and concomitant cell division (see, for example, Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed. (Pergamom Press, New York, 1990)). These agents, which include alkylating agents, such as nitrosourea, anti-metabolites, such as methotrexate and hydroxyurea, and other agents, such as etoposides, campathecins, bleomycin, doxorubicin, daunorubicin, etc., although not necessarily cell cycle specific, kill cells during S phase because of their effect on DNA replication. Other agents, specifically colchicine and the vinca alkaloids, such as vinblastine and vincristine, interfere with microtubule assembly resulting in mitotic arrest. Chemotherapy protocols generally involve administration of a combination of chemotherapeutic agents to increase the efficacy of treatment.
Despite the availability of a variety of chemotherapeutic agents, chemotherapy has many drawbacks (see, for example, Stockdale, 1998, “Principles Of Cancer Patient Management” in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10). Almost all chemotherapeutic agents are toxic, and chemotherapy causes significant, and often dangerous, side effects, including severe nausea, bone marrow depression, immunosuppression, etc. Additionally, even with administration of combinations of chemotherapeutic agents, many tumor cells are resistant or develop resistance to the chemotherapeutic agents. In fact, those cells resistant to the particular chemotherapeutic agents used in the treatment protocol often prove to be resistant to other drugs, even those agents that act by mechanisms different from the mechanisms of action of the drugs used in the specific treatment; this phenomenon is termed pleiotropic drug or multidrug resistance. Thus, because of drug resistance, many cancers prove refractory to standard chemotherapeutic treatment protocols.
There is a significant need for alternative cancer treatments, particularly for treatment of cancer that has proved refractory to standard cancer treatments, such as surgery, radiation therapy, chemotherapy, and hormonal therapy. Further, it is uncommon for cancer to be treated by only one method. Thus, there is a need for development of new therapeutic agents for the treatment of cancer and new, more effective, therapy combinations for the treatment of cancer.
There is also a clear need for cancer chemotherapeutics or therapeutic regimens for treating cancer patients while reducing or avoiding the toxicities and/or side effects associated with conventional therapies.
Citations or identification of any reference in Section 2 of this application is not to be construed that such reference is prior art to the present application.